CN101676300B - Antibody for inhibitting growth of colorectal carcinoma and its use in preparation of diagnostic agent of Colorectal cancer, endometrial carcinoma and pancreatic cancer - Google Patents
Antibody for inhibitting growth of colorectal carcinoma and its use in preparation of diagnostic agent of Colorectal cancer, endometrial carcinoma and pancreatic cancer Download PDFInfo
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- CN101676300B CN101676300B CN2008102224162A CN200810222416A CN101676300B CN 101676300 B CN101676300 B CN 101676300B CN 2008102224162 A CN2008102224162 A CN 2008102224162A CN 200810222416 A CN200810222416 A CN 200810222416A CN 101676300 B CN101676300 B CN 101676300B
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- 230000004614 tumor growth Effects 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
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Abstract
The invention provides a monoclonal antibody, which has the following characteristics: (1) the antibody is IgMs immunoglobulin and the light-chain type of the antibody is k-typed; (2) the antigen is identified into a sugar-chain structure with sialylation mucoprotein surface; the monoclonal antibody can specifically identify colorectal adenocarcinoma, endometrial adenocarcinoma and pancreatic carcinoma tissues, can not identify the corresponding normal tissues. Specifically, the monoclonal antibody is formed by the secreting of hybridomas cells with the accession number of CGMCC No. 2643, the amino acid sequence in the heavy-chain variable zone is shown as SEQ ID NO: 1, the amino acid sequence in the light-chain variable zone is shown as SEQ ID NO: 2. The invention also provides a use of the monoclonal antibody in the preparation of a diagnostic agent or a kit, wherein the diagnostic agent or the kit can be used for the screening of cancer, early-stage diagnosis, treatment monitoring and tumour imaging. The invention provides a new tool for the accurate early-stage diagnosis of colorectal cancer, endometrium and pancreatic carcinoma.
Description
Technical field
The invention belongs to the biological medicine technology field; Particularly the present invention provides specific recognition knot rectal adenocarcinoma, adenocarcinoma of endometrium and pancreatic cancer cell carbohydrate antigen; And can suppress the monoclonal antibody of colon tumor cell growth and secrete the hybridoma of this antibody, and said antibody is used for the application of the diagnostic reagent of early diagnosis colorectal cancer, carcinoma of endometrium and carcinoma of the pancreas in preparation.
Background technology
Colorectal cancer refers to the pernicious change of large intestine intimal epithelium cell, comprises unusual differentiation, abnormality proliferation and cellular metabolism and diacrisis etc.Overwhelming majority's (> 95% of colorectal cancer clinically) be gland cancer, the colorectal cancer of other type seldom comprises lymphoma, innocent tumor, pernicious sarcoma etc.The occurrence cause of knot rectal adenocarcinoma, diagnoses and treatment is preclinical medicine and clinical medical research emphasis with prevention always.
The mortality ratio of colorectal cancer occupies second of tumor mortality.The case that the U.S. is diagnosed as colorectal cancer every year surpasses 130,000, and is dead greater than 50,000, mortality ratio about 40%.Colorectal cancer is the 3rd a common digestive tract tumor in China, be positioned at after the esophageal carcinoma and the cancer of the stomach, but the annual case of dying from colorectal cancer is estimated to account for tumour 5.8% above 40,000 people.China's evident characteristic of colorectal cancer morbidity is the average age of onset one's mid-40s, shifts to an earlier date 12-18 than states such as America and Europes, and wherein the rectum cancer is more, accounts for 60%.Recent two decades, along with the progressively raising of Economic Growth in China and people's living standard, the number of the infected of China's colorectal cancer increases year by year, and morbidity more is tending towards rejuvenation.Clinical study is for many years found; If can early discovery diagnose with accurate; Nearly all colorectal cancer can both be treated (Sanduleanu and Stockbrugger, 2003) to a certain extent, and therefore effective clinical means is to reducing the dead significant of colorectal cancer.
Carcinoma of the pancreas is the high digestive tract tumor of grade malignancy, and its mortality ratio accounts for the 6th of China's malignant tumour, is positioned at the 4th in the U.S..In recent years, the sickness rate of carcinoma of the pancreas is in rising trend, and five year survival rate is still very low, and the median survival interval after diagnosis is 4～6 months (Coppola, 2000).Carcinoma of the pancreas is because of its unique anatomical position, and symptom concealment, early diagnosis are very difficult, still do not have effective method of early diagnosis at present.At present, be used for clinical tumour antigen and detect, be nonspecific taa, can only carry out joint-detection, improve the susceptibility and the validity of detection through selecting suitable tumor markers.
Carcinoma of endometrium is the malignant tumour that betides endometrial one group of epithelium property, claims carcinoma of uterine body again.Gland cancer to derive from endometrial gland is the most common, is called adenocarcinoma of endometrium.Carcinoma of endometrium is the female genital tract common cancer, and sickness rate accounts for female genital tract malignant tumour 20%～30%, and is continuous ascendant trend, accounts for women's general tumour 7%.In the treatment of Endometrial Carcinomas, operation is accepted by world wide as first-selected treatment; Postoperative chemotherapy comes into one's own gradually; But its early stage diagnosis and treatment still are current subject matter.Traditional diagnostic method is according to clinical manifestation, and auxiliary examination is analysis-by-synthesis as a result, can make a definite diagnosis like the inspection that internal film tissue is learned.
Glycosylated change is the universals of cancer cells, and has the sugar chain structure of some types to be acknowledged as the mark that tumour takes place, develops, and specific tumor markers is detected purpose (Ho et al., 1988 that can reach the early diagnosis tumour; Kim, 1990; Kim, 1998a).Digestive tube intimal epithelium cell often causes when cancerating that glycosylation changes, and one of main change is the over-expresses of Saliva Orthana (Mucin) in tumour, and another change is mucinous incomplete glycosylation.Existing research data shows that the incomplete glycosylated result of tumour cell produces Tn, T and sialic acid Tn (S-Tn) antigen, these antigenic structures under normal circumstances rarely found (Boland et al., 1986; Itzkowitz et al., 1989; Bresalier et al., 1991; Itzkowitz et al., 1992; Kim, 1998b; Hara et al., 2000).Although the pathological effect mechanism of the sugar chain of glycosylation and change in tumour generation and evolution is not clear; But TS sugar chain structure has become the affinity tag of tumor monitoring; Be used to find the early stage change of tumour; Tumor development and prognosis, the oncotherapy effect assessment, even can become the target of oncotherapy.
Although colorectal cancer detect very difficulty, at present for the diagnosis of colorectal carcinoma method of different times differ (Zhang et al., 2002; Winawer, 2005).Traditional inspection method has the intestines mirror, barivm meal fluoroscopy (screem) and intestinal contents detection etc., and diagnosis often relies on the clinician according to check result, the deduction of making in conjunction with practical experience.And for the infantile tumour that is confined to mucous membrane; Traditional detection method is difficult to make accurately to be judged; Need the sensitive detection means, set up multiple now in the molecular level detection technique, like RT-PCR; Dna probe detects and special method (Finkelstein et al., 1996a such as tumour cell markers tests; Finkelstein et al., 1996b; Hammel and Soussi, 2000; Lassmann et al., 2002), can carry out early diagnosis to colorectal cancer delicately.Yet sensitive molecular detection technology RT-PCR, dna probe detect but and to hang down in clinical application lessly because of specificity, and in-vitro diagnosis mainly is the detection that relies on the kinds of tumors affinity tag at present, and Comprehensive analysis results is judged again.
The diagnosis of colorectal cancer at present still adopts traditional inspection method to combine clinical symptom to confirm; To be existing tumor marker antibody exist factors such as poor specificity and identification antigen is uncertain to the identification of tumour to major cause; Cause the susceptibility and the specificity low (Qin Xiaoguang, 1995) that detect colorectal cancer.In most bibliographical information, monoclonal antibody is respectively 30-60% and 20-50% to the susceptibility and the specificity of colorectal cancer.CEA; CA19-9, antigenic monoclonal antibody such as CA72-4 is to the auxiliary diagnosis of colorectal cancer, like anti-CEA monoclonal antibody identification CEACAMS; Because most of tumours are in various degree expression CEA all, so anti-CEA monoclonal antibody is used to diagnose the poor specificity of a certain tumour.But for improving the recall rate of colorectal cancer, multiple clinically monoclonal antibody coupling can improve and detects positive rate and see table 1, becomes selection (Wu Daohong, 2004 of diagnosing tumour thus; Xu Xiaohong, 1998).
Table 1 CA-724, CA19-9, CEA and the positive findings of CA-50 to different tumours
At present, the diagnosis of carcinoma of the pancreas mainly realizes through the joint-detection of some tumor markerses commonly used, like CA19-9, and CA50, indexs such as CA242, they take place for diagnosing tumour, and vital role has been played in monitoring prognosis and recurrence.At present the most frequently used is with CEA, CA19-9 and CA242 coupling.The susceptibility of CA19-9 in carcinoma of the pancreas is (80%) the most by force, but specificity very low (43%) can be increased to 92% with specificity after CEA and the CA242 coupling, but after three's coupling, sensitivity is merely 29% (Ni et al., 2005).The specificity of these indexs and susceptibility still can not meet clinical needs, and therefore need to seek new diagnosis and unite the early diagnostic rate that diagnosis index further improves carcinoma of the pancreas.
In recent years, in the diagnosis of Endometrial Carcinomas, sugar antigen becomes development rapidly; Widely used tumor markers is mainly gp and glycolipid, and antigenic determinant is positioned on sugar chain or the pyrenoids; Their diagnosis that appears as clinical tumor brings convenience, like CA19-9, and CA72-4; CA125, (Yamazawa et al., 2005 such as CA15-3; Hareyama et al., 1996; Cherchi et al., 2002; Lo et al., 1999).The susceptibility of these sugar antigens is all between 30%～67%.CA19-9 and CA125 coupling, susceptibility can be up to 83.3%, and specificity is 87.2%, and visible joint-detection can effectively improve correct diagnosis, and the auxiliary diagnosis of carcinoma of endometrium is had certain value.
In decades, monoclonal antibody plays a significant role in the diagnosis of disease and treatment, and particularly to the antigenic disease of expression specificity, monoclonal antibody and antigenic reaction detection can be used as " gold " standard of medical diagnosis on disease.But different with other non-intestinal canal tumours is that colorectal cancer usually excessive secretion is expressed the glycosylated Saliva Orthana of surface elevation; Cover Saliva Orthana owing to form " sugar quilt "; The monoclonal antibody of recognition protein epitope receives sterically hindered the influence, can not effectively bring into play keying action, thereby use is little in diagnosis; On the contrary, can discern, combine the monoclonal antibody of mucin carbohydrate to become the important tool of diagnosis and treatment colorectal cancer.
Monoclonal antibody called after 3P9 provided by the invention, through detecting the selective binding of monoclonal antibody to colon cancer cell, and with normal colon cell debond characteristic, multi-turns screen obtains.Different with present clinical application IgG antibody-like, 3P9 is an IgM class monoclonal antibody, therefore has antibody structure, biochemical characteristic and the biological function that is different from IgG.Our 3P9 monoclonal antibody molecule amount that experiment showed, is greater than 600KD, and identification, to combine target epitope be the s-Tn structure, and the specific recognition colorectal cancer born of the same parents that attenuate can suppress the migration of colon cancer cell and cause the colon cancer cell apoptosis.The immunohistochemical staining of this antibody of use can be diagnosed the colorectal cancer of various differentiation degrees comparatively exactly separately; Carcinoma of endometrium; The canceration degree of carcinoma of the pancreas is confirmed cancerous region, changes to use this type of cancer susceptibility of multiple antibody combined diagnosis and/or the low present situation of specificity at present.The 3P9 monoclonal antibody suppresses the colon tumor growth and causes apoptotic function, demonstrates 3P9 monoclonal antibody potential using value aspect the treatment of adenocarcinoma of colon.
Summary of the invention
The purpose of this invention is to provide a kind of monoclonal antibody, said monoclonal antibody can combine with the cancer antigen specificity, thereby can be used to prepare the diagnostic reagent or the test kit of diagnosing cancer.
Particularly, the present invention provides following:
1. monoclonal antibody, said antibody has feature: 1) antibody is the IgM immunoglobulin like protein, light chain of antibody type κ type; 2) identification antigen is Saliva Orthana surface saliva acidifying sugar chain structure.In one embodiment, said saliva acidifying sugar chain structure is s-Tn (sialic acid N-acetylglactoside) sugar chain structure.
2. above 1 described monoclonal antibody, its specific recognition knot rectal adenocarcinoma, adenocarcinoma of endometrium and carcinoma of the pancreas tissue, the corresponding healthy tissues of nonrecognition.
3. above 1 described monoclonal antibody, it is the hybridoma cell line secretion of CGMCC No.2643 by deposit number.
4. above 1 described monoclonal antibody, its weight chain variable region amino acid sequence such as SEQ ID NO:1 demonstration, its light-chain amino acid sequence such as SEQ ID NO:2 demonstration.
5. genetic engineering antibody; Itself and above 3 or 4 described monoclonal antibodies have 30% above homologous sequence, comprising the Fab fragment, and F (ab) ' fragment; The Fd fragment; Fv fragment and Fc fragment comprise that also each fragment with homologous sequence makes up each other, or utilize part to have each fragment and other albumen of homologous sequence, the verivate of peptide chain formation.
One kind the coding above each described monoclonal antibody of 1-5 nucleotide sequence.
7. any one described monoclonal antibody is used for the diagnostic reagent of in-vivo diagnostic or in-vitro diagnosis tumour or the application of test kit in preparation in above 1 to 5.
8. above 7 described application; Wherein in-vitro diagnosis comprises: the direct or indirect above-described monoclonal antibody of mark; Utilize enzyme linked immunological absorption experiment (ELISA), immune colloidal gold technique, immunohistochemical staining; Immunofluorescence, the corresponding diagnostic method that the Western-blot detection means is set up.
9. above 7 described application; Wherein in-vivo diagnostic comprises: the direct or indirect above-described monoclonal antibody of mark; Inject or take said traget antibody, said antibody of foundation and tumour antigen association reaction carry out the location of tumor tissues and cell, tumor imaging; Tumour takes place and the development diagnosis, and keep watch on and the tumor-killing effect assessment treatment back.
10. above 7 described application, wherein said tumour are selected from the group of being made up of the tumour of expressing the sialic acid sugar chain structure.In one embodiment, said tumour is selected from by thyroid carcinoma, mammary cancer, bladder cancer cancer of the stomach, skin carcinoma, lung cancer, head and neck cancer, ovarian cancer, spermary cancer, carcinoma of the pancreas, esophagus cancer, the group that knot rectal adenocarcinoma and adenocarcinoma of endometrium are formed.In one embodiment, said tumour is selected from by carcinoma of the pancreas, the group that knot rectal adenocarcinoma and adenocarcinoma of endometrium are formed.
11. a diagnostic reagent, it is used for diagnosing tumour, comprises each described monoclonal antibody of above 1-5 as activeconstituents.In one embodiment, said tumour is colorectal cancer, carcinoma of endometrium and carcinoma of the pancreas.
12. a test kit, it is used for diagnosing tumour, comprises each described monoclonal antibody of above 1-5.In one embodiment, said tumour is colorectal cancer, carcinoma of endometrium and carcinoma of the pancreas.
The present invention uses human colon cancer cell to be antigen; Obtain the hybridoma cell strain (CGMCC No.2643) that a plant height is expressed the IgM antibody-like through immune mouse; Biochemical analysis through to this antibody is identified with the combination epi-position, confirms the glycosylated sugar chain structure of antibody capable specific recognition Saliva Orthana provided by the invention.Contrast immunohistochemical stainings normal to 213 examples and 519 routine tumor tissue sections are analyzed, and knot rectal adenocarcinoma, adenocarcinoma of endometrium and the carcinoma of the pancreas tissue and the cell of the various differentiation degrees of this antibody specific recognition are to corresponding healthy tissues nonrecognition.Therefore, this antibody can be used for tying the early diagnosis of rectal adenocarcinoma, adenocarcinoma of endometrium and carcinoma of the pancreas, treatment monitoring, examination and tumor imaging.
Description of drawings
Fig. 1 .3P9 monoclonal antibody type is identified.A. detect the antibody classification and the light chain type absorbance value of 3P9 monoclonal antibody; B. typical IgM type immunoglobulin structure synoptic diagram.
Fig. 2 .3P9 Purification of Monoclonal Antibodies and biochemical characteristic.A. through the purifying collection of illustrative plates of prepacked column Sephadex-200 chromatography, peak A, B, C, D, E are respectively the elution peak under the different elution volumes; B. dot blot (Dot blotting) detects antibody purification in the elution peak, and the result shows that antibody mainly concentrates on first peak (A); C. the native SDS-PAGE electrophorogram of antibody purification; D. the reductibility SDS-PAGE electrophoresis result figure of antibody purification.
The association reaction of Fig. 3 .3P9 monoclonal antibody and GenBank AF178428-derived protein GI 7682468 (BSM).
Fig. 4. the sodium periodate oxidation is to the influence of monoclonal antibody 3P9 and GenBank AF178428-derived protein GI 7682468 association reaction.
Fig. 5. neuraminidase is to the influence of monoclonal antibody 3P9 and GenBank AF178428-derived protein GI 7682468 association reaction.
Fig. 6. trypsinase is to the influence of monoclonal antibody 3P9 and GenBank AF178428-derived protein GI 7682468 association reaction.
Fig. 7. monoclonal antibody B72.3 combines with the competition of monoclonal antibody 3P9.
Fig. 8 .3P9 monoclonal antibody identification people ties the rectal adenocarcinoma histocyte.Monoclonal antibody 3P9 and knot rectal adenocarcinoma bonded colour attaching area mainly on epithelioglandular tenuigenin of colorectal cancer and cytolemma, also have fill the air painted simultaneously in lumen of gland; And in normal knot rectal tissue, do not have positive a chrominance signal.Arrow is depicted as the positive coloring site of yellowish brown.
Fig. 9 .3P9 monoclonal antibody identification people adenocarcinoma of endometrium histocyte.Monoclonal antibody 3P9 and adenocarcinoma of endometrium bonded colour attaching area mainly on its epithelioglandular tenuigenin and cytolemma, also have fill the air painted simultaneously in lumen of gland; And in normal endometrial tissue, do not have positive a chrominance signal.Arrow is depicted as the positive coloring site of yellowish brown.
Figure 10 .3P9 monoclonal antibody identification human pancreas cancer histocyte.Monoclonal antibody 3P9 and carcinoma of the pancreas bonded colour attaching area mainly on its epithelioglandular tenuigenin and cytolemma, also have fill the air painted simultaneously in lumen of gland; And in normal pancreatic tissue, do not have positive a chrominance signal.Arrow is depicted as the positive coloring site of yellowish brown.
Figure 11 .3P9 monoclonal antibody suppresses the migration of colon tumor cell.A-G is for adding 100ug/ml respectively, 50ug/ml, 25ug/ml; 12.5ug/ml; 6.3ug/ml 3ug/ml, 0ug/ml serial dilution monoclonal antibody 3P9 handled former generation colorectal carcinoma transplanted tumor cell after HCT-820 hour; The migration of cut district cell all is suppressed, and this restraining effect presents dose-dependently; H is cut district cell growing state before cultivating.
Figure 12 .3P9 monoclonal antibody weight chain variable region amino acid sequence and light-chain amino acid sequence.SEQ.ID.NO:1.3P9 monoclonal antibody weight chain variable region amino acid sequence; SEQ.ID.NO:2.3P9 monoclonal antibody light-chain amino acid sequence.
The cell strain 3P9 of the stably express monoclonal antibody that will obtain through colony screening is preserved in the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms on August 27th, 2008; China; Chaoyang District Beijing Da Tun road), preserving number is CGMCC No.2643.
Embodiment
Hereinafter is described reference implementation example and accompanying drawing in detail the present invention, and said embodiment only is intended as illustrative explanation the present invention, rather than intention restriction scope of the present invention.Scope of the present invention is specifically limited accompanying Claim.
The foundation of embodiment one, the strain of 3P9 monoclonal antibody hybridoma cell and the generation of monoclonal antibody
Material: 1. cell SW1116 colon carcinoma cell line, available from U.S. ATCC (American TypeCulture Collection, ATCC No.CCL-233), SP2/0 murine myeloma cell (ATCC No.CRL-1581).2. substratum serum-free DMEM; High sugared DMEM; HAT; The HT substratum is available from Gibco company; Foetal calf serum is available from Wuhan Sanli Ltd., and Tissue Culture Plate is that Coming product 3. reagent gather second
pure PEG (MW4000) available from Sigma company, and other reagent is homemade analytical pure 4. animal BALB/c mouses available from Institute of Experimental Animals, Chinese Academy of Medical Sciences.5. antibody is identified and is used mouse monoclonal antibody somatotype reagent Sigma ImmunoType Kit antibody subtype detection kit.
Method: 1. mouse immune is with 1 * 10
7/ ml SW1116 cell suspension and each 0.5ml of Fu Shi Freund's complete adjuvant (Sigma) mix emulsification, an abdominal injection 0.2ml/ mouse; 2 week backs are with the mixing and emulsifying suspension second immunisatioies of incomplete freund adjuvant (Sigma), after two weeks with the direct tail vein injection booster immunization of the cell suspension that does not contain adjuvant, the spleen isolated lymphocytes is taken out in the last immunity after 3 days.About 0.2-0.5ml serum free medium DMEM swells it to the spleen injection, punctures the spleen film with a bent injection needle multiple spot again, with the method for pushing lymphocyte is therefrom overflowed.
2. the foundation of hybridoma cell strain by 1:5 mixes centrifugal with above-mentioned mouse immune splenocyte murine myeloma cell sp2/0; Adding 1ml polyoxyethylene glycol PEG merges in 37 ℃ of water-baths; Add and to move into the T75 culturing bottle after 15ml contains the high sugared DMEM of 15% foetal calf serum; Fused cell is overnight cultures in 37 ℃, 5% CO2gas incubator, adds the HAT substratum, and making cell concn is 1 * 10
6/ ml divides cell suspension to go into 37 ℃ of two 96 orifice plates (0.2ml/ hole), 5% CO2gas incubator cultivation 3 days, changes liquid with HT substratum half amount, and 1 all backs are with the HT culture medium culturing; Select to have the culture hole of clonal growth after 7-20 days, have or not antibody expression in the detection supernatant, the method (Yan et al., 2003) of utilization enzyme linked immunological absorption (ELISA) filters out the antibody expression positive colony.
3.-the continuous clone hybridization oncocyte of the subclone of positive hybridoma cell employing limiting dilution assay (doubling dilution) filters out the cell strain of stably express antibody for 3 times, and is frozen in liquid nitrogen.
Adopt mouse peritoneal injection hybridoma to collect the method preparation of ascites 4.-ascites antibody prepares, method is referring to document (Mathews et al., 1980; Rammohan et al., 1983).
Result: the cell strain 3P9 that obtains a strain stably express monoclonal antibody through colony screening; Said cell strain is preserved in the common micro-organisms center (CGMCC of China Committee for Culture Collection of Microorganisms on August 27th, 2008; China; Chaoyang District Beijing Da Tun road), preserving number is CGMCCNo.2643.
Anti-body contg 100-200ng/ml in the cells and supernatant, anti-body contg 2-5mg/ml in the ascites.
Mouse monoclonal antibody somatotype reagent identifies that the monoclonal antibody hypotype is the IgM antibody-like, and light chain of antibody is κ type (Fig. 1), explains that this cell strain can stablize high expression level IgM/ κ type monoclonal antibody.
Embodiment two, 3P9 Purification of Monoclonal Antibodies and biochemical characteristic
Material: 3P9 monoclonal antibody ascites is obtained by hybridoma abdominal cavity inoculation BALB/c mouse results ascites; Prepackage chromatography column Sephadex-200; Column volume 100ml, the Phamacia product, horseradish peroxidase (HRP) sheep anti mouse IgM antibody is available from Sant Cruz company; The ECL fluorescence detection reagent kit uses AKTA FPLC protein chromatographic system purifying available from Pierce company.
Method: above-mentioned ascites is thawed for 4 ℃, 15, centrifugal 20 minutes of 000g collects supernatant, the degerming of 0.2um film; PH7.2,0.01M PBS balance prepacked column, ascites 5ml goes up appearance; PH7.2,0.01M PBS wash-out flow velocity 1ml/ minute, is collected each elution peak; Elution peak sample 10ul point is done dot blot (Dot blotting) and is identified on nitrocellulose filter behind the natural air drying; The albumen biochemical characteristic is with nativeSDS-PAGE and reductibility SDS-PAGE electrophoretic analysis; Biological activity of albumen is measured with Westernblotting.
Dot blot: 5% skimmed milk room temperature sealing 1 hour; PBST washing three times, again with PBS washing three times, 1:2000 dilution horseradish peroxidase (HRP) sheep anti mouse IgM antibody incubated at room 1 hour; Add enhanced chemiluminescence agent (enhanced chemiluminescence, ECL.) colour developing.
The result: ascites is through Sephadex-200 following five peaks of wash-out (Fig. 2 A), and the first peak area is maximum, accounts for 30% total peak area.Dot blot result (Fig. 2 B) shows that the function antibody component mainly is present in first peak, the second peak content pettiness, and all the other are the foreign protein peak.The first peak albumen nativeSDS-PAGE electrophoresis result (Fig. 2 C) of collecting is through Qaulity One software analysis, and purity of protein reaches 95%; Reductibility SDS-PAGE electrophoresis result (Fig. 2 D) is shown as about 75KD protein band.
Embodiment three, 3P9 monoclonal antibody combine with GenBank AF178428-derived protein GI 7682468
Material: the 3P9 monoclonal antibody is obtained through the Sephadex-200 purifying by ascites antibody; Purifying GenBank AF178428-derived protein GI 7682468 (BSM, bovine submaxillary mucin) (Sigma), horseradish peroxidase (HRP) sheep anti mouse IgM antibody is available from SantCruz company; 96 hole enzyme-linked immunosorbent assay plates are available from U.S. Corning Costar company.
Method: coating buffer dilution BSM to 50ng/ml, each plate hole adds 100ul, and room temperature was spent the night in 2 hours or 4 ℃; Get rid of coating buffer, PBS washing, 2%BSA-PBS sealing 1 hour; The 37 ℃ of incubations of 3P9 monoclonal antibody 1 hour that add 50ul serial dilution purifying, PBS or PBST washing add 50ul horseradish peroxidase (HRP) sheep anti mouse IgM antibody (PBS2; 000 times of dilution) 37 ℃ of incubations are 1 hour, and PBS washing or PBST washing add 200ul colour developing liquid (prescription: the 0.2M disodium phosphate soln of 5.1ml; Add the 0.1M citric acid solution of 4.9ml, add 10 μ l30% hydrogen peroxide and 4mg O-Phenylene Diamine (OPD)) color development at room temperature 10min, add 20 μ l2M H
2SO
4Termination reaction, 490nm detects absorbance value down.
Result: when being the antibody purification mensuration of 100ng/ml, be obvious logarithm decline (Fig. 3) behind about 0.55, the 2 times of serial dilution of absorption value with the initial concentration.Purifying 3P9 monoclonal antibody shows that with the BSM binding curve association reaction is relevant with AC, meets antigen-antibody and combines to dissociate characteristic.
Embodiment four, sodium periodate inhibition 3P9 monoclonal antibody combine with GenBank AF178428-derived protein GI 7682468
Material: the 3P9 monoclonal antibody is obtained through the Sephadex-200 purifying by ascites antibody; Purifying GenBank AF178428-derived protein GI 7682468 (BSM, bovine submaxillary mucin) (Sigma), sodium periodate is available from Sigma company, horseradish peroxidase (HRP) sheep anti mouse IgM antibody is available from Sant Cruz company; 96 hole enzyme-linked immunosorbent assay plates are available from U.S. Coming Costar company.
Method: coating buffer (15mM yellow soda ash, 35mM sodium hydrogencarbonate) dilution BSM to 50ng/ml, each plate hole adds 100ul; Room temperature was spent the night in 2 hours or 4 ℃, got rid of coating buffer, the PBS washing; Add 100ul serial dilution sodium periodate room temperature and put 20 minutes, abandon sodium periodate solution and add in the 200ul coating buffer and 5 minutes the PBS washing; Add 2%BSA-PBS sealing 1 hour, add 37 ℃ of incubations of 50ul purifying 3P9 monoclonal antibody (200ng/ml) 1 hour, PBS washing or PBST washing; Add 37 ℃ of incubations of 50ul horseradish peroxidase (HRP) sheep anti mouse IgM antibody (PBS2,000 times of dilution) 1 hour, PBS washing or PBST washing; Add 200ul colour developing liquid (prescription: the 0.2M disodium phosphate soln of 5.1ml; Add the 0.1M citric acid solution of 4.9ml, add 10 μ l30% hydrogen peroxide and 4mg O-Phenylene Diamine (OPD)) color development at room temperature 10min, add 20 μ l2M H
2SO
4Termination reaction, 490nm detects absorbance value down.
The result: after sodium periodate was handled, absorbance value increased with sodium periodate dosage and reduces, and shows obvious dose-dependence.Handle absorbance value behind the antigen less than 0.3 (Fig. 4) greater than the 1ug sodium periodate, the oxygenizement that periodates is described has influenced antibody and has combined with BSM.Because periodates can cause the sugar chain structure and the conformational change on Saliva Orthana surface, and does not influence peptide chain structure, so the epi-position structure of 3P9 antibodies Saliva Orthana BSM is sugar chain but not peptide chain.
Embodiment five, neuraminidase inhibition 3P9 monoclonal antibody combine with GenBank AF178428-derived protein GI 7682468
Material: ascites obtains purifying 3P9 monoclonal antibody through the Sephadex-200 purifying; Freeze-drying purifying GenBank AF178428-derived protein GI 7682468 (BSM, bovine submaxillary mucin) (Sigma), neuraminidase is available from Sigma company, horseradish peroxidase (HRP) sheep anti mouse IgM antibody is available from Sant Cruz company; 96 hole enzyme-linked immunosorbent assay plates are available from U.S. Coming Costar company.
Method: coating buffer dilution BSM to 50ng/ml, each plate hole adds 100ul, and room temperature was spent the night in 2 hours or 4 ℃; Get rid of coating buffer, PBS washing three times, the neuraminic acid enzyme solution (PBS preparation) that adds 100ul1ug/ml and 0.1ug/ml was respectively put 30 minutes for 37 ℃; Abandon the neuraminic acid enzyme solution and add 1M EDTA (YD 30) solution 200ul room temperature storing 5 minutes, the PBS washing adds 2%BSA-PBS sealing 1 hour; Add 37 ℃ of incubations of 50ul purifying 3P9 monoclonal antibody (200ng/ml) 1 hour, PBS washing or PBST washing add 50ul horseradish peroxidase (HRP) sheep anti mouse IgM antibody (PBS2; 000 times of dilution) 37 ℃ of incubations are 1 hour; PBS washing or PBST washing add 200ul colour developing liquid (prescription: the 0.2M disodium phosphate soln of 5.1ml, the 0.1M citric acid solution of adding 4.9ml; Add 10 μ l30% hydrogen peroxide and 4mg O-Phenylene Diamine (OPD)) color development at room temperature 10min, add 20 μ l 2M H
2SO
4Termination reaction, 490nm detects absorbance value down.
Result: after 10ng and the processing of 100ng neuraminidase; Treatment group absorbance value is had obvious reduction (Fig. 5) than control group; Further specifying the 3P9 monoclonal antibody, combine epi-position with BSM be the sugar chain part; And relevant with saliva acidifying sugar chain structure, neuraminidase to the change of sugar chain suppressed antibody to the identification of epitope with combine.
The not influence of trypsinize that combines of embodiment six, 3P9 monoclonal antibody and GenBank AF178428-derived protein GI 7682468
Material: the 3P9 monoclonal antibody is obtained through the Sephadex-200 purifying by ascites antibody; Purifying GenBank AF178428-derived protein GI 7682468 (BSM, bovine submaxillary mucin) (Sigma), trypsinase is available from Sigma company, horseradish peroxidase (HRP) sheep anti mouse IgM antibody is available from Sant Cruz company; 96 hole enzyme-linked immunosorbent assay plates are available from Coming Costar company.
Method: coating buffer dilution BSM to 50ng/ml, each plate hole adds 100ul, and room temperature was spent the night in 2 hours or 4 ℃, got rid of coating buffer; The PBS washing adds 100ul series gradient trypsinase, puts 30 minutes for 37 ℃; Abandon trypsin solution and add 1M EDTA solution 200ul room temperature storing 5 minutes, the PBS washing adds 2%BSA-PBS sealing 1 hour; Add 37 ℃ of incubations of 50ul purifying 3P9 monoclonal antibody (200ng/ml) 1 hour, PBS washing or PBST washing add 50ul horseradish peroxidase (HRP) sheep anti mouse IgM antibody (PBS2; 000 times of dilution) 37 ℃ of incubations are 1 hour, and PBS washing or PBST washing add 200ul colour developing liquid (prescription: the 0.2M disodium phosphate soln of 5.1ml; Add the 0.1M citric acid solution of 4.9ml, add 10 μ l30% hydrogen peroxide and 4mg O-Phenylene Diamine (OPD)) color development at room temperature 10min, add 20 μ l2M H
2SO
4Termination reaction, 490nm detects absorbance value down.
Result: after the gradient trypsin treatment; Except that each treatment group absorbance value has the small reduction than untreated fish group, the absorbance value considerable change does not appear, and each is organized absorption value and all is higher than 0.7; Indifference (Fig. 6) between each group; The result shows that trypsinase does not influence 3P9 monoclonal antibody and BSM association reaction to the change of Saliva Orthana peptide chain, proves antibody recognition, and the bonded epitope is non-peptide chain structure.
Embodiment seven, monoclonal antibody B72.3 competition 3P9 monoclonal antibody combine with GenBank AF178428-derived protein GI 7682468
Material: the 3P9 monoclonal antibody is obtained through the Sephadex-200 purifying by ascites antibody; Purifying GenBank AF178428-derived protein GI 7682468 (BSM, bovine submaxillary mucin) (Sigma), monoclonal antibody B72.3 working fluid is available from company of middle China fir Golden Bridge, horseradish peroxidase (HRP) sheep anti-mouse igg antibody is available from Sigma company; 96 hole enzyme-linked immunosorbent assay plates are available from Coming Costar company.
Method: monoclonal antibody B72.3 working fluid 200ul mixes (starting point concentration of 3P9 monoclonal antibody is 600ng/ml) with the 3P9 monoclonal antibody that the gradient amount increases, and each gradient is supplied 500ul with diluent (PBS), and is evenly mixed.Coating buffer dilution BSM to 50ng/ml, each plate hole adds 100ul, and room temperature was spent the night in 2 hours or 4 ℃; Get rid of coating buffer, the PBS washing adds the above-mentioned serial monoclonal antibody mixed solution of 100ul; Room temperature was put 1 hour, and the PBS washing adds 2%BSA-PBS sealing 1 hour; Add 37 ℃ of incubations of 50ul purifying 3P9 monoclonal antibody (200ng/ml) 1 hour, PBS washing or PBST washing add 50ul horseradish peroxidase (HRP) sheep anti-mouse igg antibody (PBS2; 000 times of dilution) 37 ℃ of incubations are 1 hour, and PBS washing or PBST washing add 200ul colour developing liquid (prescription: the 0.2M disodium phosphate soln of 5.1ml; Add the 0.1M citric acid solution of 4.9ml, add 10 μ l30% hydrogen peroxide and 4mg O-Phenylene Diamine (OPD)) color development at room temperature 10min, add 20 μ l2 M H
2SO
4Termination reaction, 490nm detects absorbance value down.
Result: monoclonal antibody B72.3 has specificity to combine with purifying BSM; With the increase of monoclonal antibody 3P9 amount, monoclonal antibody B72.3 combines with BSM obviously to reduce (Fig. 7), shows monoclonal antibody 3P9 competition conjugated antigen epi-position, confirms that further epitope is s-Tn (sialic acid N-acetylglactoside) sugar chain structure.
Embodiment eight, immunohistochemical analysis 3P9 monoclonal antibody are to the antigenic identification of people's tumor tissues
Material: the many tissue cancers of high-density and health adult tissue's chip (article No.: CC00-08-001) available from Shaanxi Chaoying Biotechnology Co., Ltd..It comprises 500 tissue samples from 495 routine patients, has included 12 kinds of modal cancer types of the mankind, comprises colorectal carcinoma, carcinoma of endometrium, carcinoma of the pancreas; Mammary cancer, cancer of the stomach, liver cancer, lung cancer, kidney; Skin carcinoma, head and neck cancer, ovarian cancer, spermary cancer; Bladder cancer, the cancer of the brain, prostate cancer, thyroid carcinoma etc.Every kind of cancer types comprises about 25 cancerous issue samples and 5 healthy tissues samples.The diameter of each tissue sample is 0.6mm, thickness 5 μ m (details are seen this product description).Other sample comprises human colon carcinoma tissue (52 example); Rectum cancer tissue's (50 example), uterine endometrium cancerous tissue (6 example), esophagus cancer (25 example) and corresponding healthy tissues comprise normal people colon (48 example); Rectal tissue (50 example); Uterine endometrium cancerous tissue (3 example) is taken from molecular weight tumor National Key Laboratory of Tumar Inst of Tumoor Hospital, Chinese Academy of Medical Sciences, the Chinese People's Liberation Army 301 and 306 hospitals, Beijing legal medical expert institute.Horseradish peroxidase (HRP) sheep anti mouse IgM antibody is available from Sant Cruz company; (PIN: ZLI-9033), other reagent is available from Beijing chemical reagents corporation available from company of middle China fir Golden Bridge for DAB colouring reagents box.Method: cryopreserved tissue thaws the back with 4% Paraformaldehyde 96 fixedly 16-24 hour.Experiment material after fixing is put into 70% alcohol, changes 70% alcohol once after 1 hour, through ascending gradient (80%, 90%, 100%) dehydration of alcohol, carries out the routine paraffin wax embedding, the section of 5 μ m thickness.The concrete steps of preparation paraffin organization section are referring to Zhao Zongjiang " histocyte molecules experimental principle and method ", China Traditional Chinese Medicine Publishing House,, first version in 2003.Immunohistochemical staining adopts ordinary method: section is through YLENE dewaxing, descending gradient (100%, 90%, 80%, 70%, 50%, 30%) alcohol rehydration; The room temperature black out is hatched the interference of removing endogenous peroxydase in 30 minutes in the methyl alcohol that contains 0.3% hydrogen peroxide; PBS flushing three times, each 5 minutes; Place 10mM sodium citrate buffer solution (pH6.0) to boil 10 minutes section, to repair antigen; Let section naturally cooling in damping fluid, then with PBS flushing three times, each 5 minutes; 5% sheep blood serum (China fir Golden Bridge in Beijing, PBS dilutes sheep blood serum) sealing 1 hour; Discard confining liquid; Cover the experimental group section with an anti-diluent (dilution of 1:1000 confining liquid) that contains the 3P9 monoclonal antibody; The control group section still covers with 5% sheep blood serum, with containing two underlined anti-(the sheep anti mouse IgM of horseradish peroxidase, Santa Cruz companies; The dilution of 1:500 confining liquid) cover experimental group and control group section, 37 hatched 1 hour; PBS flushing three times, each 5 minutes; DAB colouring reagents box with fresh configuration develops the color at last; Microscopically is observed and is developed the color to appropriate level, the reaction of PBS flushing color development stopping; Redye section 1-2 minute with hematoxylin solution; Tap water flushing section 10 minutes; Ascending gradient alcohol (30%, 50%, 70%, 80%, 90%, 100%) dehydration, the transparent back of YLENE resin mounting; Observe after the section of 37 ℃ of drying in oven and take pictures.
The result: the 3P9 monoclonal antibody is to the positive rate difference of various cancer types very big (table 2), and combination degree also has very big difference.Wherein the recall rate to adenocarcinoma of colon is the highest, reaches 71.8%, secondly is adenocarcinoma of endometrium (68%), rectal adenocarcinoma (62%), and carcinoma of the pancreas (55.6%), and the strongest to the combination degree of these cancers.
Table 2. people tumor tissues 3P9 Detection of antigen.
1.3P9 monoclonal antibody is tied the antigenic identification of rectal adenocarcinoma histocyte to the people
Monoclonal antibody 3P9 identification, combination knot rectal adenocarcinoma cell and secretory product thereof; Appear strong yellowish brown (such as arrow among Fig. 8 demonstration) coupling reaction; And healthy tissues does not have coupling reaction (Fig. 8); Instruction book clonal antibody 3P9 can specific recognition, combine the people to tie the product of rectal adenocarcinoma cell and secreting, expressing, i.e. antigen.Immunohistochemical methods interpretation of result to 125 routine clinical adenocarcinoma of colon tissues and healthy tissues shows; Monoclonal antibody 3P9 identification human colon adenocarcinoma's susceptibility 71.8%; (51/71); Specificity 79.6% (43/54), positive predictive value (PPV) 82.3% (51/62), negative predictive value (NPV) 68.3% (43/63); Immunohistochemical methods interpretation of result to 100 routine clinical rectal adenocarcinoma tissues and healthy tissues shows; The susceptibility 62% of monoclonal antibody 3P9 identification people rectal adenocarcinoma; (31/50); Specificity 62% (31/50), positive predictive value (PPV) 62% (31/50), negative predictive value (NPV) 62% (31/50) is seen table 3.
Table 3.3P9 monoclonal antibody detects knot rectal adenocarcinoma, adenocarcinoma of endometrium, the susceptibility of carcinoma of the pancreas and specificity analyses.
PPV: positive predictive value; NPV: negative predictive value;
2.3P9 monoclonal antibody is to the antigenic identification of people's adenocarcinoma of endometrium histocyte
Monoclonal antibody 3P9 identification, combination uterine endometrium adenocarcinoma cell and secretory product thereof; Appear strong yellowish brown coupling reaction (such as arrow among Fig. 9 demonstration); And healthy tissues does not have coupling reaction (Fig. 9), and instruction book clonal antibody 3P9 can discern, combine the product of people's endometrial gland cancer cells and secreting, expressing.Immunohistochemical methods interpretation of result to 37 routine clinical endometrial gland cancerous tissues and healthy tissues shows; The susceptibility 68% (17/25) of monoclonal antibody 3P9 identification people adenocarcinoma of endometrium; Specificity 100% (12/12); Positive predictive value (PPV) 100% (17/17), negative predictive value (NPV) 60% (12/20) is seen table 3.
3.3P9 monoclonal antibody is to the antigenic identification of human pancreas cancer histocyte
Monoclonal antibody 3P9 identification, combination pancreatic cancer cell and secretory product thereof; Appear strong yellowish brown coupling reaction (such as arrow among Figure 10 demonstration); And healthy tissues does not have coupling reaction (Figure 10), and instruction book clonal antibody 3P9 can discern, combine the product of human pancreatic cancer cell and secreting, expressing.Immunohistochemical methods interpretation of result to 25 routine clinical carcinoma of the pancreas tissues and healthy tissues shows; The susceptibility 55.6% of monoclonal antibody 3P9 identification human pancreas cancer; (10/18); Specificity 100% (7/7), positive predictive value (PPV) 100% (10/10), negative predictive value (NPV) 46.7% (7/15) is seen table 3.
4.3P9 monoclonal antibody is to the relation of the positive rate and the tumour differentiation degree of various cancers
Immunohistochemical methods is the result show, 3P9 has the downward trend with the reduction of tumour differentiation degree to the recall rate of various cancers, and this is particularly evident in adenocarcinoma of endometrium and the carcinoma of the pancreas at adenocarcinoma of colon.But pass through χ
2Check the difference of positive rate between the different differentiation degrees of these cancers, find not significant difference (table 4), instruction book clonal antibody 3P9 does not have dependency to the positive rate of various cancers and the differentiation degree of tumour.
The relation of table 4.3P9 Detection of antigen positive rate and tumour differentiation degree.
(-) do not have positive staining or positive cell number proportion less than 10%; (+) positive cell number proportion is more than or equal to 10% but less than 25%; (++) positive cell number proportion is more than or equal to 25% but less than 50%; (+++) positive cell number proportion is more than or equal to 50%; NS does not have significant difference; UND, unknown differentiation degree.
Embodiment nine, 3P9 monoclonal antibody suppress the migration of tumour cell
Material: colorectal carcinoma transplanted tumor in nude mice HCT-8 is provided by animal housing of Tumar Inst of Tumoor Hospital, Chinese Academy of Medical Sciences, trypsinase, and collagenase I is available from Sigma company; Tissue Culture Plate is Falcon (Becton Dickinson.Labware, Franklin Lakes, NJ; USA) product, cell culture fluid are that DMEM (contains 10% foetal calf serum, penicillium mould 100U/ml; Streptomycin sulphate 50ug/ml), all the other reagent are available from Beijing chemical company.
Method: the colorectal carcinoma transplanted tumor in nude mice is cut into 1mm
3Fritter prepares former generation colorectal carcinoma transplanted tumor cell HCT-8 with reference to " Zooblast culture medium present technique guide " (Science Press, R.I. Fu Leixieni,, the 4th edition in 2000) method.When the HCT-8 cell grows into individual layer,, abandon nutrient solution with liquid-transfering gun head cut on monolayer; PBS washes cell twice; Add the DMEM nutrient solution that contains serial dilution monoclonal antibody 3P9, conventional cell cultures 20 hours is observed cut district cell growing state under phase microscope.
The result: compare with cultivating preceding cut (Figure 11 H), monoclonal antibody 3P9 suppresses the migration of HCT-8 colon cancer cell of former generation, and restraining effect presents dose-dependently (Figure 11 A-G).
Reference
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Sequence table
< 110>Institute of Biophysics, Academia Sinica
< 120>novel antibody of a kind of human colon tumor of inhibition growth and be used for the application of the diagnostic reagent of early diagnosis colorectal cancer, carcinoma of endometrium and carcinoma of the pancreas in preparation
<130>IB085793
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<170>PatentIn?version3.1
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< 221>3P9 monoclonal antibody light-chain amino acid sequence
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Claims (8)
1. monoclonal antibody, said antibody has feature: 1) antibody is the IgM immunoglobulin like protein, light chain of antibody type κ type; 2) identification antigen is Saliva Orthana surface saliva acidifying sugar chain structure; Said monoclonal antibody specific recognition knot rectal adenocarcinoma, adenocarcinoma of endometrium and carcinoma of the pancreas tissue; The corresponding healthy tissues of nonrecognition, it is the hybridoma cell line secretion of CGMCC No.2643 by deposit number.
2. the described monoclonal antibody of claim 1, its weight chain variable region amino acid sequence such as SEQ ID NO:1 demonstration, its light-chain amino acid sequence such as SEQ ID NO:2 demonstration.
3. nucleotide sequence of claim 1 or 2 described monoclonal antibodies of encoding.
4. claim 1 or 2 described monoclonal antibodies are used for the diagnostic reagent of in-vivo diagnostic or in-vitro diagnosis tumour or the application of test kit in preparation, and wherein said tumour is selected from by carcinoma of the pancreas, the group that knot rectal adenocarcinoma and adenocarcinoma of endometrium are formed.
5. the described application of claim 4; Wherein in-vitro diagnosis comprises: direct or indirect mark claim 1 or 2 described monoclonal antibodies; Utilize enzyme-linked immunosorbent assay (ELISA); Immune colloidal gold technique, immunohistochemical staining, the corresponding diagnostic method that immunofluorescence or Western-blot detection means are set up.
6. the described application of claim 4, wherein in-vivo diagnostic comprises: direct or indirect mark claim 1 or 2 described monoclonal antibodies, inject or take said traget antibody; Said antibody of foundation and tumour antigen association reaction carry out the location of tumor tissues and cell, tumor imaging, and tumour takes place and the development diagnosis; Keep watch on and the tumor-killing effect assessment treatment back; Wherein said tumour is selected from by carcinoma of the pancreas, the group that knot rectal adenocarcinoma and adenocarcinoma of endometrium are formed.
7. diagnostic reagent, it is used for diagnosing tumour, comprises claim 1 or 2 described monoclonal antibodies as activeconstituents, and wherein said tumour is selected from by colorectal cancer, the group that carcinoma of endometrium and carcinoma of the pancreas are formed.
8. test kit, it is used for diagnosing tumour, comprises claim 1 or 2 described monoclonal antibodies, and wherein said tumour is selected from by colorectal cancer, the group that carcinoma of endometrium and carcinoma of the pancreas are formed.
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| CN86100512A (en) * | 1986-02-21 | 1987-09-02 | 国家医药管理局上海医药工业研究院 | 2 of antitumor action is arranged, and 4-diamino-6-(N-methyl or formyl radical-replacement benzyl amino) quinoline derivatives is synthetic |
| CN101676301A (en) * | 2008-09-16 | 2010-03-24 | 中国科学院生物物理研究所 | Antibody for inhibitting growth of colorectal carcinoma and its use in preparation of medicament and kit |
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| CN86100512A (en) * | 1986-02-21 | 1987-09-02 | 国家医药管理局上海医药工业研究院 | 2 of antitumor action is arranged, and 4-diamino-6-(N-methyl or formyl radical-replacement benzyl amino) quinoline derivatives is synthetic |
| CN101676301A (en) * | 2008-09-16 | 2010-03-24 | 中国科学院生物物理研究所 | Antibody for inhibitting growth of colorectal carcinoma and its use in preparation of medicament and kit |
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